In recent years, there has been renewed emphasis on folate-based therapeutics for cancer, reflecting on capacities for tumor-selective membrane transport. This application explores the therapeutic potential for the human proton-coupled folate transporter (hPCFT). Unlike the human reduced folate carrier (hRFC), the major tissue folate transporter which is ubiquitously expressed and has a neutral pH optimum, hPCFT shows a narrower tissue distribution and is characterized by an acid pH optimum. hPCFT shows substantial transport at acid pHs characterizing the tumor microenvironment and limited transport at neutral pH. We showed that hPCFT is widely expressed in human tumor cell lines and primary tumors. In human tumor cells, hPCFT transcripts paralleled levels of hPCFT proteins on Western blots and transport activity. In human tumor cells, hPCFT reporter gene activity paralleled disparate levels of hPCFT transcripts. We initiated hPCFT structure-function studies and characterized a mechanistically and structurally important re-entrant loop domain between transmembrane domains (TMDs) 2 and 3 and localized a substrate binding region within TMD2. We demonstrated a functionally important role for hPCFT oligomerization and localized an important oligomer interface to TMD6. In a separate line of study, we identified novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates that are selectively transported by hPCFT but not hRFC, although uptake by folate receptor was also preserved. These efforts have fostered a new paradigm, the rational development of tumor-targeted therapies based on tumor-specific expression and/or function of the major folate transporters, PCFT and FR. We will continue our comprehensive study of the major facilitative folate transporters, with implications to folate-based therapies for cancer, and to prophylactic folate supple- mentation for cancer. Our emphasis is on optimizing our novel hPCFT-selective therapeutics for tumor targeting, by exploring the biology of this physiologically important transporter, drawing from a compelling rationale for targeting solid tumors by this mechanism. In Specific Aim 1, we will characterize molecular and cellular determinants of hPCFT- targeted cancer therapy, including establishing functional stoichiometries for hRFC vis a vis hPCFT, the therapeutic impact of concomitant tumor expression of hPCFT and hRFC, as well as folate receptor , and mechanisms of hPCFT transcriptional regulation in human tumor cells. In Aim 2, we will characterize structural and functional determinants of hPCFT-targeted therapy, including identification of functionally important domains and residues in the hPCFT protein, and the structural, functional, and regulatory features of hPCFT homo-oligomers. The goal is to use biochemical studies of hPCFT structure and function, and molecular homology modeling, to assist our drug discovery efforts, and to identify new approaches to enhance tumor targeting and efficacy of hPCFT-selective drugs. Our proposed studies are strongly supported by our preliminary and published results and the availability of critical reagents and cell lines, includig the first generation of specific and potent hPCFT-selective tumor-targeted antifolates that are not transported by hRFC. Our studies are distinctive for their novelty and likelihood of providing critical new insights into mechanisms and regulation of these physiologically and pharmacologically important vitamin transporters that can be translated to the clinic.